The performance of an immunoassay in the detection of a specified analyte is expressed in terms of sensitivity and specificity. The former is a measure of the test to correctly identify the samples in a population that are positive for the analyte. The latter is a measure of the test to correctly identify samples in a population that do not contain the analyte.
Immunoassay performance may be adversely affected by a number of interfering factors. Alleviation of these interferences may be effected by methods that are directed toward their cause. For example, as is described by Yolken (Yolken, R. H., 1982, Rev. Infec. Dis., 4:35-68) and is well established in the art, certain generic additives to assay reagents tend to improve the performance of the assay. Detergents such as Triton X-100 and proteins such as bovine serum albumin (BSA) reduce nonspecific protein-protein interactions and, thus, minimize non-immunochemical reactivities. Also, chelating agents such as ethylenediaminetetraacetate (EDTA) may increase the efficiency of added detergents and minimize complementmediated phenomena, thereby improving immunoassay performance. Changes in assay performance brought about by the present invention were shown to be additional to the effects of detergents, nonspecific proteins and chelating agents.
Certain substances may be involved in specific but undesirable reactivities that occur within the chemistry of an immunoassay. Improvements in assay performance brought about by this invention do not rely upon the addition or subtraction of any new chemical or antigen. In an ELISA using a single, unconjugated antigen, the solid phase would already be coated with antigen and blocked with BSA. It is not obvious that replacement of a portion of the unconjugated antigen with BSA-conjugated antigen would constitute a chemical change in the character of the solid phase.
Some antigens may undergo changes in conformation, and therefore changes in reactivity, as a result of binding to carriers or solid surfaces (McCullough, K. C., Crowther, J. R. and Butcher, R. N., 1985, J. Immunol. Meth., 82:91-100). This phenomenon has been shown to be responsible for differences in sensitivity and specificity between immunoassay formats using the same reagents. Also, pH and ionic strength have been shown to affect the solid phase adsorption characteristics of proteins (Geerligs, H. J. et al., 1988, J. Immunol. Meth., 106:239-244). These factors would not be expected to be of importance in assays where highly specific binding partners are utilized as the binding and detection reagents. In the specific situation where one of the binding partners is a monoclonal antibody, a change in the conformation of an antigen to which the antibody is highly reactive would be expected to result in decreased rather than increased assay performance.
The chemistry by which reagents are bound to a solid phase may affect the performance of the resulting assay. Solid phase preparation protocols include passive, chemical, biochemical, immunologic (Brennand, D. M., Danson, M. J. and Hough, D. W., 1986, J. Immunol. Meth., 93:9-14) and covalent (Geerligs, H. J. et al., 1988) binding methods.
Certain antigens do not bind well to certain solid phase materials. It is not uncommon to conjugate those antigens to a carrier reagent such as, but not limited to, hemocyanin (Geerlings, H. J. et al., 1988) or BSA (Skurrie, I. J. and Gilbert, G. L., 1983, J. Clin. Microbiol., 17, 738-43) that will enhance their immobilization. This tends to increase the sensitivity of the subsequent assay by providing a solid phase that has more antigen available for reactivity with an incoming antibody. Other proteins such as ovalbumin, gelatin, casein, to name a few, can also be used as carriers. The improvement in assay performance that is brought about with the use of this invention relies upon a combination of conjugated and unconjugated antigen forms. Sensitivity and specificity are compromised by the use of either antigen form alone.
Jitsukawa et al., 1989, (Jitsukawa, T., Nakajima, S., Sugawara, I., and Watanabe, H., 1989, J. Immunol. Meth., 116:251-7) report an improvement in assay performance as a result of simultaneously binding a physical mixture of antigen and BSA or other "effector protein" to immunoassay plates. These authors state specifically that the observed results were due to independent binding of the two agents to the solid phase and not to each other. Their method is a one step coating and blocking procedure and does not result in the creation of a solid phase that is fundamentally different from those of established protocols.
A competition immunoassay for the detection of antibody directed against hepatitis B core antigen (HBcAg) has been described (Adamich, M. and Wos, S. M., U.S. Pat. No. 4,818,688, Apr. 4, 1989), incorporated here by reference. This assay format, using a high-affinity monoclonal antibody and purified, unconjugated antigen, is the state of the art technology over which the improvements comprising the present invention are based.